Flagella are important structural elements determining the motility of bacteria and are generally composed of a hook, a basal body and a filament. It is known that flagella contribute to the swimming or swarming motility, the taxis of bacteria, the adhesion of pathogenic microorganisms to host cells, and formation of biofilms. A subunit protein forming the flagellar filament is referred as flagellin, and flagellins are regularly assembled to form the filament. Hayashi et al. reported that TLR5 expressed on mammalian cells recognize the flagellin of gram-negative and gram-positive bacteria to activate NF-κB (Hayashi F, Smith K D, Ozinsky A, Hawn T R, Yi E C, Goodlett D R, Eng J K, Akira S, Underhill D M, Aderem A: Nature 410:1099-1103, 2001).
Toll-like receptors (TLRs) are typical “Pattern Recognition Receptors (PRRs)”, which recognize “Pathogen Associated Molecular Patterns (PAMPs)” present in pathogens and are found not only in mammals, but also on the surfaces of insects and plant cells. Thirteen kinds of TLRs have been found to date, and studies on agonists of TLRs have been actively conducted (Akira S, Uematsu S, Takeuchi O: Cell 124 (4): 783-801, 2006).
PRRs such as TLRs are distributed on the surface or in the cytoplasm of host cells, induce innate immune responses after being stimulate with various PAMPs, and furthermore, regulate adaptive immune responses. Thus, TLR agonists can serve targets suitable for the development of various immune regulators, particularly vaccine adjuvants.
As used herein, the term “vaccine adjuvants” refers to substances which can enhance, prolong or accelerate Ag-specific immune responses induced by vaccine antigens, when they are co-administered with vaccines. Vaccine adjuvants approved for use in the human body include aluminum phosphate, aluminum hydroxide, and squalene emulsion. Vaccine adjuvants must satisfy at least one of the following five requirements: 1) regulation of expression of co-stimulatory molecules on the surface of antigen-presenting cells, induction of antigen-specific T-lymphocyte responses, or immunomodulation such as the modulation of cytokine secretion; 2) antigen presentation; 3) induction of CD8+ cytotoxic T lymphocyte responses; 4) targeting; and 5) depot generation.
Ideal vaccine adjuvants: 1) must be safe; 2) must be biodegraded in vivo; 3) must show potent protective or therapeutic immune responses compared to when antigens are administered alone; 4) must be chemically or biologically verified substances; 5) preferably act at concentrations lower than antigens; and 6) must have a long half life, such that they can be readily applied commercially or clinically.
Substances, which are currently used as vaccine adjuvants or considered for use as vaccine adjuvants, include: 1) mineral salts such as aluminum hydroxide gel; 2) surfactant substances; 3) bacteria-derived substances; 4) cytokines or hormones; 5) polyanions; 6) polyacryl; 7) carriers; 8) living vectors comprising virus; and 9) vehicles, such as mineral oil liposomes. Among them, protein-derived vaccine adjuvants, which are currently actively studied and receive great attention, include Vibrio cholerae-derived cholera toxin (CT) and Escherichia coli-derived heat-labile toxin (LT). It was reported that these vaccine adjuvants induce the production of antigen-specific antibodies in mucosal areas and sera and induce the expression of B7-2 on the surface of antigen-presenting cells (APCs) to stimulate the co-stimulatory signaling of CD4+ helper T cells. However, these adjuvants are exotoxins having high enterotoxicity, and thus studies focused on reducing the toxicity thereof and increasing the adjuvanticity thereof, are in progress.
As disclosed in PCT International Patent Publication No. WO 2005/070455, the present inventors constructed a transposon mutant library in order to screen adhesion and invasion factors of Vibrio vulnificus, and analyzed the Tn-flanking regions in three clones, which lost their adhesion to host cells and their motility, and, as a result, the present inventors identified two flagella operons consisting of 56 genes. In this analysis process, it was found that V. vulnificus has a total of six flagellin genes (flaA, flaB, flaF, flaC, flaD and flaE), and among them, the flaB gene is the major component of the flagellins. The present inventors studied the possibility that the flagellin recombinant protein (FlaB), the component of the polar flagellin of V. vulnificus, can be applied as a component vaccine against V. vulnificus, and, as a result, the present inventors found that the flagellin recombinant protein (FlaB), besides high antigenicity, also has a strong vaccine adjuvant effect. To elucidate this finding, the present inventors conducted further studies. As a result, it was demonstrated that, when a vaccine antigen tetanus toxoid was administered to the nasal cavity of test animals together with flagellin, the flagellin amplified the effect of the vaccine by transmitting a signal to the TLR5 of host cells to activate the immune system, and when a lethal dose of tetanus toxin was challenged to mice immunized by administering tetanus toxoid and flagellin, the flagellin induced complete defense immunity to the toxin, suggesting that the flagellin had an excellent mucosal vaccine adjuvant effect (Lee S E, Kim S Y, Jeong B C, Kim Y R, Bae S J, Ahn O S, Lee J J, Song H C, Kim J M, Choy H E, Chung S S, Kweon M N, Rhee J H.: Infect. Immun. 74: 694-702, 2006).
Among various TLR agonists, flagellin stimulating TLR5 is a protein component, unlike other TLR agonists [CpG-DNA, MLP (mycoplasmal lipopeptide)]. Thus, it is possible to synthesize recombinant flagellin proteins, the quality of which can be continuously controlled, and in addition, it is possible to construct various recombinant fusion proteins having an enhanced activity of stimulating TLR5.
According to the results of studies on the three-dimensional structure of flagellin, polar amino acid residues and charged amino acid residues in a flagellin monomer react with those in another monomer, so that the axial interaction between the monomers occurs to form a polymer, thus forming the characteristic filament structure of flagellin (Yonekura K, Maki-Yonekura S, Namba K: Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature. 2003 424(6949):643-50; Samatey F A, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410(6826):331-7). According to the study of Smith et al., it was reported that TLR5 does not recognize a filament-type flagellin polymer, but recognizes flagellin monomers (Smith K D, Andersen-Nissen E, Hayashi F, Strobe K, Bergman M A, Barrett S L, Cookson B T, Aderem A. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat. Immunol. 2003 4(12):1247-53).
A common problem, observed in preventive and therapeutic vaccines (vaccines for infectious diseases, autoimmune diseases, allergic diseases and cancers), which are currently used in clinical applications, is that these vaccines lack effective vaccine adjuvants, which can specifically amplify relevant responses. Thus, there is a strong need to develop safer and more effective vaccine adjuvants.